This is an application for an exploratory research grant to identify new forms of the hepatitis C virus (HCV) non- structural proteins produced in infected human liver tissue and to develop models for their formation and function. HCV continues to be a significant public-health concern of global proportion. In spite of years of effot, the inability to study the numerous genotypes of HCV in cell culture remains an obstacle to our understanding of the mechanisms employed by this virus to establish persistence, which can ultimately lead to the development of hepatocellular carcinoma (HCC). HCV non-structural protein 5A (NS5A) is unique among non-structural proteins of most positive-strand RNA viruses. Two of its three domains are intrinsically disordered, and it contains numerous sites of phosphorylation for myriad serine/threonine kinases, including cAMP-activated protein kinase (PKA), casein kinase 1 family members and casein kinase 2. We have been intrigued by the possibility that specific patterns of NS5A phosphorylation produce unique conformations and functions for this protein, thus explaining the ability of NS5A to interact with so many cellular proteins and pathways. Our experimental tests of this possibility have led to the identification of the site of NS5A phosphorylation by PKA and to the development of immunological reagents to demonstrate the existence of the PKA-phosphorylated form of NS5A in cells replicating HCV RNA of both genotypes 1b and 2a. In addition, examination of biopsies from pediatric cases of hepatitis C and tissue from an end-stage case of hepatitis C, led to the discovery that forms of NS5A observed in infected liver tissue are different than those observed in human hepatoma cell lines replicating HCV RNA. The additional forms of NS5A detected in liver tissue are remarkably similar to those observed in cell lines when caspases are activated and the abundance of the PKA- phosphorylated species correlates well with the amount of liver injury. It is our hypothesis that caspase cleavage of NS5A could provide a mechanism for HCV to monitor the antiviral state of the hepatocyte; phosphorylation of NS5A could signal to HCV that an anti-apoptotic state suitable for replication exists in the hepatocyte. These cleaved and phosphorylated forms may expand the NS5A proteome, creating forms of the protein essential for virus multiplication. If this is the case, then the inability of most HCV sequences to replicat in hepatoma cell lines may be related, in part, to the inability to produce forms of NS5A, and perhaps other non-structural proteins, that are readily produced in hepatocytes and that are required for optimal virus multiplication. Our working model will be explored by pursuing the following specific aims: (1) Identification and characterization of forms of HCV non-structural proteins produced during infection in vivo; (2) Evaluation of pro-apoptotic and pro-survival (anti-apoptotic) responses during HCV infection in vivo; and (3) Elucidation of clinical correlations. These studies have the ability to discover forms of HCV non-structural proteins unique to the infected hepatocyte and to reveal correlations between the level of these new forms and clinical outcomes. PUBLIC HEALTH RELEVANCE: Hepatitis C virus (HCV) is a major cause of liver disease worldwide; only two HCV sequences and one cell line are available to study the HCV lifecycle in vitro. We have discovered that forms of some HCV non-structural proteins observed in HCV-infected liver are not produced in cell lines, and we propose that the absence of these forms in cell lines limits HCV multiplication in vitro. Therefore, completion of this study may reveal new approaches to study more HCV genotypes and facilitate the development of new antiviral therapies.